Microfluidic chip

ABSTRACT

A microfluidic chip orients and isolates components in a sample fluid mixture by two-step focusing, where sheath fluids compress the sample fluid mixture in a sample input channel in one direction, such that the sample fluid mixture becomes a narrower stream bounded by the sheath fluids, and by having the sheath fluids compress the sample fluid mixture in a second direction further downstream, such that the components are compressed and oriented in a selected direction to pass through an interrogation chamber in single file formation for identification and separation by various methods. The isolation mechanism utilizes external, stacked piezoelectric actuator assemblies disposed on a microfluidic chip holder, or piezoelectric actuator assemblies on-chip, so that the actuator assemblies are triggered by an electronic signal to actuate jet chambers on either side of the sample input channel, to jet selected components in the sample input channel into one of the output channels.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of, and claims prioritybenefit from, U.S. Non-provisional patent application Ser. No.14/579,441, filed on Dec. 22, 2014, which is a continuation-in-part ofU.S. Non-provisional patent application Ser. No. 13/943,322, filed onJul. 16, 2013, which issued as U.S. Pat. No. 8,961,904 on Feb. 24, 2015.The '441 and '322 applications are hereby incorporated by referenceherein, in their entireties.

The present invention relates to a microfluidic chip design which isused to isolate particles or cellular materials into various componentsand fractions, using laminar flows.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a microfluidic chip design which isused to isolate particles or cellular materials into various componentsand fractions, using laminar flows.

2. Description of the Related Art

In the separation of various particles or cellular materials—forexample, the separation of sperm into viable and motile sperm fromnon-viable or non-motile sperm, or separation by gender—the process isoften a time-consuming task, with severe volume restrictions. Thus,current separation techniques cannot, for example, produce the desiredyield, or process volumes of cellular materials in a timely fashion.

Thus, there is needed a separation technique and apparatus which iscontinuous, has high throughput, provides time saving, and which causesnegligible or minimal damage to the various components of theseparation. In addition, such an apparatus and method should havefurther applicability to biological and medical areas, not just in spermsorting, but in the separation of blood and other cellular materials,including viral, cell organelle, globular structures, colloidalsuspensions, and other biological materials.

SUMMARY OF THE INVENTION

The present invention relates to a microfluidic chip system, whichincludes a microfluidic chip loaded on a microfluidic chip cassettewhich is mounted on a microfluidic chip holder.

In one embodiment, the microfluidic chip includes a plurality of layersin which are disposed a plurality of channels including: a sample inputchannel into which a sample fluid mixture of components to be isolatedis inputted; a first plurality of sheath fluid channels into whichsheath fluids are inputted, the first plurality of sheath fluid channelswhich intersect the sample input channel at a first intersection, suchthat the sheath fluids compress the sample fluid mixture on at least twosides, such that the sample fluid mixture becomes a relatively smaller,narrower stream, bounded by the sheath fluids, while maintaining laminarflow in the sample input channel; a second plurality of sheath fluidchannels, substantially of the same dimensions as the first plurality ofsheath fluid channels, into which sheath fluids are inputted, the secondplurality of sheath fluid channels which intersect the sample inputchannel at a second intersection downstream from the first intersection,in a second direction substantially 90 degrees above and below thesample input channel, such that the sheath fluids from the secondplurality of sheath fluid channels compress the sample fluid mixture,such that the components in the sample fluid mixture are compressed andoriented in a predetermined direction, while still maintaining laminarflow in the sample input channel; and a plurality of output channelsstemming from the sample input channel, the plurality of output channelswhich removes the components and the sheath fluids from the microfluidicchip.

In one embodiment, the microfluidic chip includes an interrogationapparatus which interrogates and identifies said components in saidsample fluid mixture in said sample input channel, in an interrogationchamber disposed downstream from said second intersection.

In one embodiment, the microfluidic chip includes an isolating mechanismwhich isolates selected of said components in said sample fluid mixturedownstream from said interrogation chamber, by displacing a trajectoryof a stream of said sample fluid mixture in said sample input channel,and pushing said selected components in said displaced stream of samplefluid mixture into one of said plurality of output channels which leadfrom said interrogation chamber.

In one embodiment, the microfluidic chip further includes at least onejet chamber containing sheath fluids introduced into said jet chamber byat least one air vent; and at least one jet channel which is connectedto said at least one jet chamber, said at least one jet channel whichenters said sample input channel to said interrogation chamber.

In one embodiment, the isolating mechanism includes at least onepiezoelectric actuator assembly disposed on at least one side of saidsample input channel.

In one embodiment, the piezoelectric actuator assembly is an external,stacked piezoelectric actuator assembly.

In one embodiment, the microfluidic chip further includes a diaphragmwhich covers each said jet chamber; and wherein said external, stackedpiezoelectric actuator assembly aligns with and displaces saiddiaphragm, to drive said sheath fluids in said jet chamber into saidsample input channel, to displace said trajectory of said stream of saidsample fluid mixture in said sample input channel into one of saidplurality of output channels.

In one embodiment, the external, stacked piezoelectric actuator assemblyis disposed in a microfluidic chip holder.

In one embodiment, the microfluidic chip further includes an electroniccircuit connected to the piezoelectric actuator assembly, the electroniccircuit which amplifies an electronic signal generated by a resistanceforce from the piezoelectric actuator being in contact with thediaphragm.

In one embodiment, an electric signal from the piezoelectric film showshow much strain is generated by the external, stacked piezoelectricactuator assembly.

In one embodiment, an indicator of contact is turned on automaticallywhen contact between the piezoelectric actuator and the diaphragm ismade.

In one embodiment, when sensing of the contact is made, the electronicsignal exceeds a set threshold, and the piezoelectric actuator assemblycompresses the jet chamber to jet sheath fluids from the jet chamberinto the sample fluid channel.

In one embodiment, the indicator of contact includes a light, a sound, ahaptic, or any combination thereof.

In one embodiment, the piezoelectric actuator assembly includes aflexible diaphragm which covers said jet chamber; and a piezoelectricmaterial bonded on a top surface of said diaphragm by an adheringmechanism.

In one embodiment, when voltage is applied across electrodes of thepiezoelectric actuator assembly, the flexible diaphragm bends into thejet chamber and squeezes the sheath fluids from the jet chamber into thesample input channel to deflect the selected components into one of theplurality of output channels.

In one embodiment, the jet channel is tapered when it connects to thesample input channel.

In one embodiment, the microfluidic chip further includes a plurality ofoutputs disposed at ends of said plurality of output channels.

In one embodiment, the plurality of output channels increase indimension from the sample input channel.

In one embodiment, the microfluidic chip further includes a plurality ofnotches disposed at a bottom edge of the microfluidic chip to isolatethe plurality of outputs.

In one embodiment, the sample input channel and the plurality of sheathchannels are disposed in one or more planes of the microfluidic chip.

In one embodiment, the sample input channel and the plurality of sheathchannels are disposed in one or more structural layers, or in-betweenstructural layers of the microfluidic chip.

In one embodiment, at least one of the plurality of sheath channels isdisposed in a different plane than a plane in which the sample inputchannel is disposed.

In one embodiment, at least one of the plurality of sheath channels isdisposed in a different structural layer than a structural layer inwhich the sample input channel is disposed.

In one embodiment, the sample input channel tapers at an entry pointinto the first intersection with said plurality of sheath channels.

In one embodiment, the sample input channel tapers into saidinterrogation chamber.

In one embodiment, the plurality of sheath fluid channels taper at entrypoints into the sample input channel at least one of the firstintersection or the second intersection.

In one embodiment, the interrogation chamber includes an opening cutthrough the structural layers in the microfluidic chip; and a top windowis configured to receive a first covering in an opening in at least onelayer of the structural layers; and a bottom window is configured toreceive a second covering in an opening in at least one layer of thestructural layers.

In one embodiment, the interrogation chamber includes an opening cutthrough the planes in the microfluidic chip; and a top window isconfigured to receive a first covering in an opening in at least oneplane of the planes of the microfluidic chip; and a bottom window isconfigured to receive a second covering in an opening in at least oneplane of the planes of the microfluidic chip.

In one embodiment, the interrogation apparatus includes a light sourceconfigured to emit a beam through the first covering, to illuminate andexcite the components in said sample fluid mixture; and wherein emittedlight induced by the beam passes through said second covering and isreceived by an objective lens.

In one embodiment, the interrogation apparatus includes a light sourceconfigured to emit a beam through structural layers of the microfluidicchip, to illuminate and excite the components in the sample fluidmixture; and wherein emitted light induced by the beam is received by anobjective lens.

In one embodiment, the interrogation apparatus includes a light sourceconfigured to emit a beam through the planes of the microfluidic chip,to illuminate and excite said components in said sample fluid mixture;and wherein emitted light induced by said beam is received by anobjective lens.

In one embodiment, the emitted light received by the objective lens isconverted into an electronic signal which triggers said piezoelectricactuator assembly.

In one embodiment, one of the sample fluid mixture or the sheath fluidsis pumped into the microfluidic chip by a pumping apparatus.

In one embodiment, the external tubing communicates fluids to themicrofluidic chip.

In one embodiment, the components are cells.

In one embodiment, wherein the cells to be isolated include at least oneof viable and motile sperm from non-viable or non-motile sperm; spermisolated by gender and other sex sorting variations; stem cells isolatedfrom cells in a population; one or more labeled cells isolated fromun-labeled cells including sperm cells; cells, including sperm cells,distinguished by desirable or undesirable traits; genes isolated innuclear DNA according to a specified characteristic; cells isolatedbased on surface markers; cells isolated based on membrane integrity orviability; cells isolated based on potential or predicted reproductivestatus; cells isolated based on an ability to survive freezing; cellsisolated from contaminants or debris; healthy cells isolated fromdamaged cells; red blood cells isolated from white blood cells andplatelets in a plasma mixture; or any cells isolated from any othercellular components into corresponding fractions.

In one embodiment, the isolated components are moved into one of theplurality of output channels, and unselected components flow out throughanother of the plurality of output channels.

In one embodiment, the microfluidic chip further includes a computerwhich controls the pumping of one of the sample fluid mixture or thesheath fluids into the microfluidic chip.

In one embodiment, the microfluidic chip further includes a computerwhich displays the components in a field of view acquired by a CCDcamera disposed over the opening in the microfluidic chip.

In one embodiment, the microfluidic chip system, includes: amicrofluidic chip loaded on a microfluidic chip cassette which ismounted on a microfluidic chip holder, the microfluidic chip having asample input for introducing sample fluid into the microfluidic chip,and sheath inputs for introducing sheath fluid into the microfluidicchip; and a pumping mechanism which pumps said sample fluid from areservoir into the sample input of the microfluidic chip, and pumps thesheath fluids into the sheath inputs of the microfluidic chip.

In one embodiment, a method of orienting and isolating components in afluid mixture, includes: inputting a sample fluid mixture containingcomponents into a sample input channel of a microfluidic chip; inputtingsheath fluids into a plurality of first sheath fluid channels of themicrofluidic chip, the sheath fluids from the first sheath fluidchannels which join the sample fluid mixture in the sample input channelat a first intersection of the plurality of first sheath fluid channelsand the sample input channel; wherein the sheath fluids from the firstsheath fluid channels compress the sample fluid mixture in one directionin the sample input channel to focus the components in the sample fluidmixture around a center of the sample input channel; and inputtingsheath fluids into a plurality of second sheath fluid channels of themicrofluidic chip, the sheath fluids from the plurality of second sheathchannels which join the sample fluid mixture in the sample input channelat a second intersection of the plurality of second sheath fluidchannels and the sample input channel, downstream from the firstintersection; wherein the sheath fluids from the plurality of secondsheath fluid channels further compress the sample fluid mixture at thesecond intersection, in a second direction, such that the components arefocused and aligned in a center of the sample input channel by bothwidth and depth as the components flow through the sample input channel;and wherein the sheath fluids act on the components to compress andorient the components in a selected direction as the components flowthrough the sample fluid channel.

Thus has been outlined, some features consistent with the presentinvention in order that the detailed description thereof that followsmay be better understood, and in order that the present contribution tothe art may be better appreciated. There are, of course, additionalfeatures consistent with the present invention that will be describedbelow and which will form the subject matter of the claims appendedhereto.

In this respect, before explaining at least one embodiment consistentwith the present invention in detail, it is to be understood that theinvention is not limited in its application to the details ofconstruction and to the arrangements of the components set forth in thefollowing description or illustrated in the drawings. Methods andapparatuses consistent with the present invention are capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein, as well as the abstract included below, are for thepurpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe methods and apparatuses consistent with the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects, features and advantages of the present invention will bemore readily appreciated upon reference to the following disclosure whenconsidered in conjunction with the accompanying drawings, in which:

FIG. 1 shows an exploded perspective view of an illustrative embodimentof a microfluidic chip according to one embodiment consistent with thepresent invention.

FIGS. 2A-2C show top views of the assembled microfluidic chip of FIG. 1,according to variant embodiments consistent with the present invention.

FIG. 3 shows a cross-sectional view of an interrogation chamber of themicrofluidic chip of FIGS. 1-2, according to one embodiment consistentwith the present invention.

FIG. 4 shows a cross-sectional internal view of an illustrativeinterrogation by a light source of components flowing in a fluidmixture, through the microfluidic chip of FIGS. 1-2, and an illustrativeaction of one of two (mirrored) piezoelectric actuator assemblies,according to one embodiment consistent with the present invention.

FIG. 5A shows a perspective internal, and oblique view of componentsflowing through the microfluidic chip of FIGS. 1-2, and an illustrativeoperation of two-step focusing, according to one embodiment consistentwith the present invention.

FIG. 5B shows a perspective oblique view of the channels andinterrogation chamber disposed in the microfluidic chip of FIGS. I-2C,according to one embodiment consistent with the present invention.

FIG. 6 shows a schematic illustration of a front view of a main body ofa microfluidic chip holder, according to one embodiment consistent withthe present invention.

FIG. 7 shows a schematic illustration of a side view of a piezoelectricactuator assembly of the microfluidic chip holder of FIG. 6, accordingto one embodiment consistent with the present invention.

FIG. 8 shows a schematic illustration of a front view of a microfluidicchip holder, according to one embodiment consistent with the presentinvention.

FIG. 9 shows a pumping mechanism which pumps sample fluid and sheath orbuffer fluids into the microfluidic chip, according to one embodimentconsistent with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Before turning to the figures, which illustrate the illustrativeembodiments in detail, it should be understood that the presentdisclosure is not limited to the details or methodology set forth in thedescription or illustrated in the figures. It should also be understoodthat the terminology is for the purpose of description only and shouldnot be regarded as limiting. An effort has been made to use the same orlike reference numbers throughout the drawings to refer to the same orlike parts.

The present disclosure relates to a microfluidic chip design, which isused to isolate particles or cellular materials, such as sperm, andother particles or cells, into various components and fractions, usinglaminar flows.

The various embodiments of the present invention provide for isolatingcomponents in a mixture, such as, for example: isolating viable andmotile sperm from non-viable or non-motile sperm; isolating sperm bygender, and other sex sorting variations; isolating stems cells fromcells in a population; isolating one or more labeled cells fromun-labeled cells distinguishing desirable/undesirable traits; isolatinggenes in nuclear DNA according to a specified characteristic; isolatingcells based on surface markers; isolating cells based on membraneintegrity (viability), potential or predicted reproductive status(fertility), ability to survive freezing, etc.; isolating cells fromcontaminants or debris; isolating healthy cells from damaged cells(i.e., cancerous cells) (as in bone marrow extractions); red blood cellsfrom white blood cells and platelets in a plasma mixture; and isolatingany cells from any other cellular components, into correspondingfractions.

In addition, the subject matter of the present disclosure is alsosuitable for other medical applications as well. For example, thevarious laminar flows discussed below may be utilized as part of akidney dialysis process, in which whole blood is cleansed of wasteproducts and returned to the patient. Further, the various embodimentsof the present disclosure may have further applicability to otherbiological or medical areas, such as for separations of cells, viruses,bacteria, cellular organelles or subparts, globular structures,colloidal suspensions, lipids and lipid globules, gels, immiscibleparticles, blastomeres, aggregations of cells, microorganisms, and otherbiological materials. For example, the component separation inaccordance with the present disclosure may include cell “washing”, inwhich contaminants (such as bacteria) are removed from cellularsuspensions, which may be particularly useful in medical and foodindustry applications. Significantly, prior art flow-based techniqueshave not recognized any applicability to separation of non-motilecellular components, as have the present invention.

The subject matter of the present disclosure may also be utilized tomove a species from one solution to another solution where separation byfiltering or centrifugation is not practical or desirable. In additionto the applications discussed above, additional applications includeisolating colloids of a given size from colloids of other sizes (forresearch or commercial applications), and washing particles such ascells, egg cells, etc. (effectively replacing the medium in which theyare contained and removing contaminants), or washing particles such asnanotubes from a solution of salts and surfactants with a different saltconcentration or without surfactants, for example.

The action of isolating species may rely on a number of physicalproperties of the objects or components including self-motility,self-diffusivity, free-fall velocity, or action under an external force,such as an actuator, an electromagnetic field or a holographic opticaltrap. The properties which may be sorted upon include, for example, cellmotility, cell viability, object size, object mass, object density, thetendency of objects to attract or repel one another or other objects inthe flow, object charge, object surface chemistry, and the tendency ofcertain other objects (i.e., molecules) to adhere to the object.

The various embodiments of the microfluidics chip, as described below,utilize one or more flow channels, having a plurality of substantiallylaminar flows, allowing one or more components to be interrogated foridentification, and to be isolated into flows that exit into one or moreoutputs. In addition, the various components in the mixture may beisolated on-chip by using further isolation mechanisms, such as, forexample, flow mechanisms, or optical tweezing or holographic opticaltrapping, or by magnetics (i.e., using magnetic beads). The variousembodiments of the present invention thereby provide separation ofcomponents on a continuous basis, such as, within a continuous, closedsystem without the potential damage and contamination of prior artmethods, particularly as provided in sperm separation. The continuousprocess of the present invention also provides significant time savingsin isolating components.

While discussion below focuses on the separation of sperm into viableand motile sperm from non-viable or non-motile sperm, or isolating spermby gender and other sex sorting variations, or isolating one or morelabeled cells from un-labeled cells distinguishing desirable/undesirabletraits, etc., the apparatus, methods and systems of the presentinvention may be extended to other types of particulate, biological orcellular matter, which are capable of being interrogated by fluorescencetechniques within a fluid flow, or which are capable of beingmanipulated between different fluid flows into different outputs.

While the present subject matter is discussed in detail with respect toa microfluidic chip 100 illustrated in FIGS. 1-5 and a microfluidic chipholder 200 illustrated in FIGS. 6-9, it should be understood that thisdiscussion applies equally to the various other embodiments discussedherein or any variation thereof.

Microfluidic Chip Assembly

FIG. 1 is an illustrative embodiment of a microfluidic chip 100. Themicrofluidic chip 100 is manufactured of a suitable thermoplastic (e.g.,low auto-fluorescing polymer etc.) through an embossing process orinjection molding process, as well known to one of ordinary skill in theart, and is of suitable size.

The microfluidic chip 100 includes a plurality of structural layers inwhich are disposed micro-channels which serve as sample inputchannel(s), sheath or buffer fluid channel(s), output channel(s), etc.The micro-channels are of suitable size to accommodate a particulatelaminar flow, and may be disposed in any of the layers of the chip 100in the appropriate length, as long as the object of the presentinvention is realized. The desired flow rate through the microfluidicchip 100 may be controlled by a predetermined introduction flow rateinto the chip 100, maintaining the appropriate micro-channel dimensionswithin the chip 100, by pumping mechanisms, providing narrowing of themicro-channels at various locations, and/or by providing obstacles ordividers within the micro-channels.

A plurality of inputs is provided into the microfluidic chip 100, whichprovide access to the micro-channels/channels. In one embodiment, asshown in FIGS. 1-2, a sample input 106 is used for introducing a sampleof components 160 in a sample fluid mixture 120 (see FIGS. 4-5) into asample input channel 164A of the microfluidic chip 100 from a reservoirsource (see FIG. 9). The microfluidic chip 100 also includes at leastone sheath/buffer input (in one embodiment, sheath/buffer inputs 107,108) for the introduction of sheath or buffer fluids. In one embodiment,there are two sheath/buffer inputs in the microfluidic chip 100, whichinclude a sheath/buffer input 107 and sheath/buffer input 108, bothdisposed proximate to the sample input 106, and which both introducesheath or buffer fluids into the microfluidic chip 100. The sheath orbuffer fluids are well known in the art of microfluidics, and in oneembodiment, may contain nutrients well known in the art to maintain theviability of the components 160 (i.e., sperm cells) in the fluidmixture. The location of the sheath/buffer inputs 107, 108 may vary, andthey may access micro-channels in the chip 100 which are in the same ordifferent structural layers.

In one embodiment, fill holes or air vents 121, 122—if not sealed—can beused to introduce sheath or buffer fluids into jet chambers 130, 131(described later).

In one embodiment, a plurality of output channels stemming from mainchannel 164 (see FIG. 2A) is provided for removal of fluid which hasflowed through the microfluidic chip 100, including the isolatedcomponents 160 and/or sheath or buffer fluids. In one embodiment asshown in FIGS. 1-2, there are three output channels 140-142 whichinclude a left side output channel 140, a center output channel 141, anda right side output channel 142. The left side output channel 140 endsat a first output 111, the center output channel 141 ends at a secondoutput 112 and the right side output channel 142 ends at a third output113. However, the number of outputs may be less or more depending on thenumber of components 160 to be isolated from the fluid mixture 120.

In one embodiment, instead of a straight edge, where necessary, aplurality of notches or recesses 146 are disposed at a bottom edge ofthe microfluidic chip 100 to separate the outputs (i.e., outputs111-113) and for attachment of external tubing etc. The first output111, the second output 112 and the third output 113 are reached viaoutput channels 140-142 which originate from interrogation chamber 129(see FIGS. 2A-4).

In one embodiment, the microfluidic chip 100 has a plurality ofstructural layers in which the micro-channels are disposed. The channelsmay be disposed in one or more layers or in-between layers. In oneembodiment, as shown in FIG. 1, as an example, four structural plasticlayers 101-104 are shown to comprise the microfluidic chip 100. However,one of ordinary skill in the art would know that less or additionallayers may be used, and the channels may be disposed in any of thelayers as long as the object of the present invention is achieved.

A gasket of any desired shape, or O-rings, may be provided to maintain atight seal between the microfluidic chip 100 and the microfluidic chipholder 200 (see FIG. 6). In the case of a gasket, it may be a singlesheet or a plurality of components, in any configuration, or material(i.e., rubber, silicone, etc.) as desired. In one embodiment, as shownin FIG. 1, a first gasket 105 is disposed at one end of the microfluidicchip 100 and interfaces, or is bonded (using an epoxy) with layer 104. Aplurality of holes 144 are provided in the first gasket 105 and areconfigured to align with the sample input 106, sheath/buffer input 107,sheath/buffer input 108, and air vents 121, 122, to provide accessthereto.

In one embodiment, a second gasket 143 is disposed at another end of themicrofluidic chip 100 opposite to the first gasket 105, and interfacesor is bonded with (using epoxy) the top structural layer 104. The secondgasket 143 is configured to assist sealing, as well as stabilizing orbalancing the microfluidic chip 100 in the microfluidic chip holder 200(see FIG. 6).

In one embodiment, holes and posts 145 are disposed at variousconvenient positions in the microfluidic chip 100 to fix and align themultiple layers (i.e., layers 101-104) during chip fabrication.

In one embodiment, a sample fluid mixture 120 including components 160is introduced into sample input 106, and the fluid mixture 120 flowsthrough main channel 164 toward interrogation chamber 129 (see FIGS. 2A,4, and 5). The sheath or buffer fluids 163 are introduced intosheath/buffer inputs 107, 108, and flow through channels 114, 115 and116, 117, respectively, into the main channel 164, and towards theinterrogation chamber 129 before flowing out through output channels140-142.

In one embodiment, sheath or buffer fluids 163 can be introduced intojet chambers 130, 131 through air vents 121, 122 to fill the chambers130, 131 after manufacture of the microfluidic chip 110, if the chambers130, 131 are not filled with sheath or buffer fluids 163 duringmanufacture. As stated above, the sheath or buffer fluids 163 used arewell known to one of ordinary skill in the art of microfluidics.

In one embodiment, the fluid mixture 120 from main channel 164 joinswith the sheath or buffer fluids 163 from channels 114, 115 atintersection 161 in the same plane of the microfluidic chip 100. In oneembodiment, buffer fluids 163 from channels 116, 117 join the combinedfluid mixture 120 and sheath or buffer fluids 163 from firstintersection 161, downstream at second intersection 162. In oneembodiment, channels 114, 115 are substantially the same dimensions aschannels 116, 117, as long as the desired flow rate(s) is achieved toaccomplish the object of the present invention.

In one embodiment, channels 114-117, 123, 124, 140-142, 125 a, 125 b,126 a, 126 b, 127, 128 may have substantially the same dimensions,however, one of ordinary skill in the art would know that the size ofany or all of the channels in the microfluidic chip 100 may vary indimension (i.e., between 50 and 500 microns), as long as the desiredflow rate(s) is achieved to accomplish the object of the presentinvention.

In one embodiment, the channels 114-117, 123, 124, 140-142, 125 a, 125b, 126 a, 126 b, 127,128, of the microfluidic chip 100, may not justvary in dimension, but may have tapered shapes at entry points to otherchannels in the chip 100 in order to control the flow of fluid throughthe channels. For example, main channel 164 may taper at the entry pointinto intersection 161 (see FIG. 5B), to control and speed up the flow ofsample 120 into the intersection 161, and allow the sheath or bufferfluids 163 from channels 114, 115 to compress the sample 120 fluidmixture in a first direction (i.e., horizontally), on at least twosides, if not all sides (depending on where the tapered channel 164joins channel 164A). Thus, the sample fluid mixture 120 becomes arelatively smaller, narrower stream, bounded or surrounded by sheath orbuffer fluids 163, while maintaining laminar flow in channel 164A.However, one of ordinary skill in the art would know that the mainchannel 164 entering into intersection 161 could be of any physicalarrangement, such as a rectangular or circular-shaped channel, as longas the object of the present invention is obtained.

In one exemplary embodiment, at least one of the channels 116, 117 isdisposed in a different structural layer of the microfluidic chip 100,than the layer in which the channel 164 is disposed. For example,channel 116 may be disposed in layer 103 and channel 117 may be disposedin layer 101 (see FIG. 1), such that channels 116, 117 are at differentplanes from the other channels 164 and 114, 115 (in layer 102), when thesheath or buffer fluids 163 join the fluid mixture 120 at intersection162. In one embodiment, main channel 164 is disposed between layers 102,103 (see FIG. 3); however, one of ordinary skill in the art would knowthat the channels 114-117, 164, 123, 124, 140-142, 125 a, 125 b, 126 a,126 b, 127, 128 etc., can be disposed in any layer or between any twolayers. Further, although the channels 114-117, 164, 123, 124, 140-142,125 a, 125 b, 126 a, 126 b, 127, 128 etc. are described in exemplaryembodiments as shown in the Figures, one of ordinary skill in the artwould know that the particular arrangement or layout of the channels onthe chip 100 may be in any desired arrangement as long as they achievethe described features of the present invention.

In one embodiment, the sheath or buffer fluids in channels 116, 117 jointhe fluid mixture via holes cut in the layers 101-103 at substantiallyvertical positions above and below the intersection 162. The sheath orbuffer fluids from channels 116, 117 compress the fluid mixture 120 flowin a perpendicular manner with respect to channel 164B, such that thecomponents 160 in the fluid mixture 120 are compressed or flattened, andoriented in the selected or desired direction (see below), while stillmaintaining laminar flow in channel 164B.

In one embodiment, as shown in FIGS. 1-2, channels 114, 115 and 116, 117are depicted as partially coaxial to one another with a center pointdefined by the sample input 106. Thus, in one embodiment, channels 114,115 and 116, 117 are disposed in a substantially parallel arrangement,with the channels 114, 115 and 116, 117 being equidistant to mainchannel 164. However, one of ordinary skill in the art would recognizethat the depicted configuration may be different as long as it achievesthe desired features of the present invention.

Further, in one embodiment, channels 114, 115 preferably joinintersection 161 in the same plane, at an angle of 45 degrees or less,whereas channels 116, 117, which parallel sample input channel 164A,join intersection 162 from different layers, at an angle ofsubstantially 90 degrees. However, one of ordinary skill in the artwould appreciate that the depicted configurations, angles, andstructural arrangements of the microfluidic chip 100 layers and channelsmay be different as long as they achieve the desired features of thepresent invention.

In one embodiment, downstream from intersection 162, the components 160in the fluid mixture 120 flow through channel 164B into an interrogationchamber 129, where the components 160 are interrogated.

In one embodiment, a flexible diaphragm 170, 171 (see FIG. 1) made froma suitable material, such as one of stainless steel, brass, titanium,nickel alloy, polymer, or other suitable material with desired elasticresponse, covers jet chambers 130, 131. In one embodiment, an actuatoris disposed on at least one side of channel 164B and the interrogationchamber 129 (see FIGS. 2A and 2B), in order to cause mechanicaldisplacement of the diaphragm 170, 171, in order to jet or push sheathor buffer fluids 163 from one of the jet chambers 130, 131 on that sideof channel 146B, to push components 160 from channel 164C into one ofthe output channels 140, 142 on the other side of the channel 164B. Inother words, the actuator would jet sheath or buffer fluids 163 from jetchamber 130 into channel 164C, and push target components 160 in channel164C into output channel 142 to isolate the target components from thefluid mixture 120. This embodiment is useful when only one type oftarget components 160 are isolated (which may require only two outputchannels 141, 142, for example, instead of three output channels140-142) (see FIG. 2B).

The actuator may be one of a piezoelectric, magnetic, electrostatic,hydraulic, or pneumatic type actuator. Although a disc-shaped actuatorassembly (i.e., 109, 110) is shown in FIGS. 1-2, one of ordinary skillin the art would know that any type or shape of actuator which performsthe needed function could be used.

In other embodiments, the actuator is disposed on either side of channel164B (as shown in FIG. 2A), but in other embodiments, more than oneactuator (of a relatively smaller size) may be disposed on one or moresides of channel 164 B and connected to channel 164 B via jet channels(see FIG. 2C).

The following description of the function of the actuator(s) will bemade with reference to FIG. 2A, although one of ordinary skill in theart would know that any type of actuator disposed in a location on thechip 100 would be acceptable, as long as it achieved the features of thepresent invention.

In one embodiment, in order to activate the diaphragms 170, 171 and jetthe sheath or buffer fluids 163 from chambers 130, 131 into channel164B, two external, stacked piezoelectric actuator assemblies 209, 210are provided (see FIGS. 6 and 7) which align with and actuate thediaphragms 170, 171. The external, stacked piezoelectric actuatorassemblies 209, 210 are disposed in a microfluidic chip holder 200. Thestacked piezoelectric actuator assemblies 209, 210 each include apiezoelectric actuator 219,220, respectively, which have a high resonantfrequency, and which each are disposed at a position in a center of, andin contact with the diaphragm 170, 171, to squeeze the sheath or bufferfluids 163 from the chamber 130, 131 into channel 164C.

The microfluidic chip holder 200 may be of any type known to one ofordinary skill in the art, and is configured to precisely positionpiezoelectric actuators 219, 220, such that the piezoelectric actuators219, 220 may maintain constant contact with diaphragms 170, 171 of themicrofluidic chip 100. For example, in one embodiment, this isaccomplished by each piezoelectric actuator assembly 209,210 beingmounted (or adhered using a suitable epoxy) on lockable adjustmentscrews 201 which move the piezoelectric actuators 219, 220 into positionagainst the diaphragms 170, 171, respectively; and thumb screws 202 withthreaded bodies that act to move the screws 202 against the diaphragms170, 171 for stabilization. A spacer 203 attached to the piezoelectricactuator 219,220 allows a feasible contact to be made between it and adiaphragm 170, 171 of the microfluidic chip 100. The adjustment screws201 allow a user to adjust the position of the piezoelectric actuators209, 210 relative to the microfluidic chip 100 for both coarse and fineadjustment. The thumb screws 202 may be tightened to secure thepiezoelectric assemblies 209, 210 to the main chip body 100 or loosenedto remove the piezoelectric actuator assemblies 209, 210 from the mainchip body 100.

In one embodiment, at least one piezoelectric actuator (209 or 210) ismounted on a plate (not shown) which can be translated in directionnormal to the diaphragm (170 or 171) of the microfluidic chip 100. Anadjustment screw 201 is mounted on the holder 200 and can be extendedand retracted by turning the screw 201. The tip of the adjustment screw201 is against the plate. As the screw 201 is extended, the plate alongwith the piezoelectric actuator 209, 210, is pushed toward the diaphragm170, 171 in a translation motion, such that a feasible contact is madebetween the piezoelectric actuator 209,210 and the diaphragm 170, 171.With this method, the positioning of the piezoelectric actuators 209,210 is adjusted through translation of the piezoelectric actuators 209,210 only, while in the previous embodiment where the piezoelectricactuator 209,210 is mounted directly on the adjustment screw 201, thepositioning of the piezoelectric actuator 209,210 is a combination oftranslation and rotation of the piezoelectric actuator 209,210, duringwhich damage can be caused on the delicate piezoelectric actuator209,210.

In another embodiment, an electronic circuit is connected to the stackedpiezoelectric actuator assembly 209,210 before driving it. When each ofthe stacked piezoelectric actuators 219,220 is in contact with therespective diaphragm 170, 171, the resistance force from the diaphragm170, 171 causes the strain on the stacked piezoelectric actuator219,220, which generates an electronic signal. Thus, the electroniccircuit is able to amplify the electronic signal to a predeterminedvalue to trigger an LED (light emitting diode). When the stackedpiezoelectric actuator 219,220 is in contact with the diaphragm 170, 171the LED is turned on automatically, which indicates contact between thestacked piezoelectric actuator 219,220 and the diaphragm 170, 171 ismade. This contact sensing allows enough force for the actuators 219,220 to compress the chambers 130, 131 to jet fluid 163 into the channel164B.

It would be clear to one of ordinary skill in the art that the LED isone example of an indicator of contact. For example, once contact ismade, and the electronic signal exceeds a set threshold, a feedback isgenerated for the user, which can be in any of the following forms: alight (i.e., LED), a sound (i.e., buzzer), a haptic (i.e., vibrator), orany combination thereof. Thus, the user can stop adjusting the contactand sustain the contact. Of course, in one embodiment, theabove-described procedure may be automated.

In an alternative embodiment, instead of at least one external stackedpiezoelectric actuator assembly, a thin film of piezoelectric material(well-known to one of ordinary skill in the art) is directly depositedon the top surface of at least one diaphragms 170, 171, to form at leastone piezoelectric actuator assembly 109, 110 (see FIGS. 2A and 4) todisplace (bend) the respective diaphragm 170, 171 and drive the fluidsin the respective jet chamber 130, 131 into channel 164C, respectively.The piezoelectric material is permanently bonded with the previouslydescribed flexible diaphragm 170, 171 by an adhering mechanism. Thus, inthis embodiment, when voltage is applied across the electrodes of thepiezoelectric actuator assembly 109,110, the whole diaphragm 170,171bends into the chamber 130,131 and squeezes the fluid 163 therein, intothe channel 164C to deflect the target or selected components 160 into aside output channel 140, 142.

As stated above, with respect to either the external stackedpiezoelectric actuator assemblies 209, 210 or piezoelectric actuatorassemblies 109, 110, in one embodiment, only one piezoelectric actuatorassembly may be required to jet sheath or buffer fluids 163 from jetchamber 130 into channel 164C, and push target components 160 in channel164C into output channel 142 to isolate the target components from thefluid mixture 120, as shown in FIG. 2B.

In one embodiment, the piezoelectric actuator assemblies 109, 110 areused to seal the jet chambers 130, 131, respectively, at layer 103, forexample—but one of ordinary skill in the art would know that it could bein any structural layer—after the chambers 130, 131 are filled withsheath or buffer fluids 163, to make the microfluidic chip 100impervious to fluid leakage.

Thus, the piezoelectric actuator assemblies 109, 110 satisfy therequirement of low flow rates considering the relatively small benddisplacement of the diaphragms 170, 171, and low force thereon, incontrast to the large displacement and strong force applied by theexternal, stacked piezoelectric actuator assemblies 209, 210 which areable to work at very high flow rates. However, one of ordinary skill inthe art would know that the actuator assemblies 109, 110, 209, 210 canbe chosen independently for use in the microfluidic chip 100 based uponthe different operation speeds and flow rate requirements.

In one embodiment, a thin piezoelectric film disposed on top of thediaphragm 170, 171 works as a strain sensor to determine how much strainor displacement the external, stacked piezoelectric actuator assemblies209, 210 generate as they are triggered by the electronic signal todisplace the respective diaphragms 170, 171. The diameter and thicknessof the piezoelectric thin film depends on the cross-section of theexternal, stacked piezoelectric actuator 219,220 and the force generatedon the diaphragm 170, 171. The piezoelectric thin film and diaphragm170, 171 may be different from the one discussed above in thealternative embodiment.

The filling of the jet chambers 130, 131 is now described. In oneembodiment, air vents 121,122 are provided to remove air from jetchambers 130, 131 respectively (see FIG. 2A), after manufacturing whenthe chambers 130, 131 are filled with sheath or buffer fluids163—forcing air out through the air vents 121, 122—and before thechambers 130, 131 are sealed with sheath or buffer fluids 163 therein.Alternatively, in another embodiment, if the air vents 121, 122 are leftopen, then sheath or buffer fluids 163 may be introduced through thevents 121, 122 into the chambers 130, 131 if this is not done duringmanufacturing. The sheath of buffer, or other fluids 163 disposed in thejet chambers 130, 131 may be the same or different from the sheath orbuffer fluids 163 inputted through channels 114, 115, 116, or 117.

In one embodiment, if sheath or buffer fluids 163 are used to fill upthe jet chambers 130, 131, they may be inputted through inputs 121, 122and flowed through channels 123, 124 respectively, to enter jet chamber130 via channels 125 a and 125 b, and jet chamber 131 via channels 126 aand 126 b.

In one embodiment, jet channel 127 leaves jet chamber 130, and jetchannel 128 leaves jet chamber 131, and both jet channels 127, 128 enterthe interrogation chamber 129 (see FIG. 2A). The jet channels 127, 128may be disposed in any layer of the chip 100 and enter the channel 164Cat any angle in the same plane.

In one embodiment, in order to form a strong, instantaneous jet stream,the jet channels 127, 128 may be tapered when they connect to the mainchannel 164C. However, one of ordinary skill in the art would know thatthe jet channels 127, 128 may have a particular angle, or be of adifferent structure, as long as they achieve the described features ofthe present invention.

In one embodiment, the jet channels 127, 128 work to displace or bendthe diaphragms 170, 171, respectively, and jet or squeeze sheath orbuffer fluids 163 into the channel 164C. However, when the diaphragms170, 171 return to a neutral (unbent) position, the jet channels 127,128 which issue from jet chambers 130, 131, work as diffusers to ensurethat a net fluid volume from the jet chambers 130, 131 to the channel164C is maintained, and that it is easy to refill the chambers 130, 131with sheath or buffer fluids 163.

In one embodiment, output channels 140-142 depart from channel 164Cwithin interrogation chamber 129 to outputs 111-113. As stated above, inone embodiment, more than one on-chip piezoelectric actuator assembly109, 110, or external, stacked piezoelectric actuator assembly 209,210(in any size or location) may be used to connect to each of jet channels127, 128, to provide additional power to jet sheath or buffer fluids 163from jet chambers 130, 131 into channel 164C. In one embodiment, thedistance from each of the jet channels 127, 128 entries into channel164C to each of the output channels 140-142 should be shorter than thedistance between components 160, to avoid target components 160 mixingwith undesired components 160 (further described below). In oneembodiment, the cross-section and the length of the output channels140-142 should be maintained at a predetermined volume ratio (i.e.,2:1:2, or 1:2:1 etc.) to obtain the desired hydraulic resistance of theoutput channels 140-142.

In one embodiment, an interrogation apparatus is disposed downstreamfrom where channels 116, 117 enter into channel 164B. In one embodiment,channel 164B tapers into the interrogation chamber 129, which speeds upthe flow of the fluid mixture through the interrogation chamber 129.However, one of ordinary skill in the art would know that the channel164 B need not taper and could be of any dimension and size as long asthe present invention performs according to the desired requirements.

An interrogation apparatus is used to interrogate and identify thecomponents 160 in the fluid mixture in channel 164B passing through theinterrogation chamber 129. Note that channel 164B may be disposed in asingle layer (i.e., layer 102) or disposed in between layers (i.e.,layers 102, 103). In one embodiment, the interrogation chamber 129includes an opening or window 149 (see FIG. 3) cut into the microfluidicchip 100 in at least the uppermost layer (i.e., layer 104 or other), andanother opening or window 152 is cut into the chip 110 in at least thelowermost layer (i.e., layer 101 or other).

In one embodiment, an opening 150 is cut into the microfluidic chipthrough layers 101-104. In one embodiment, the top window 149 isconfigured to receive a first covering 133 and the bottom window 152 isconfigured to receive a second covering 132. However, the windows 149,152 may be located in any suitable layer and need not be in theuppermost/lowermost layers. The coverings 133, 132 may be made of anymaterial with the desired transmission requirements, such as plastic,glass, or may even be a lens. Note that although the relative diametersof the windows 149, 152 and opening 150 are shown in FIG. 3, these mayvary according to manufacturing considerations.

In one embodiment, the above-mentioned first and second coverings 133,132 are configured to enclose the interrogation chamber 129. The windows149, 152 and coverings 133, 132 (see FIG. 3), allow the components 160flowing in the fluid mixture 120 in channel 164B (see FIG. SA) throughthe interrogation chamber 129, to be viewed through opening 150, andacted upon by a suitable light source 147 configured to emit a highintensity beam 148 with any wavelength that matches excitable componentsin the fluid mixture 120. Although a laser 147 is shown, any suitableother light sources may be used, such as a light emitting diode (LED),arc lamp, etc. to emit a beam which excites the components.

In one embodiment, a high intensity laser beam 148 from a suitable laser147 of a preselected wavelength—such as a 355 nm continuous wave (CW)(or quasi-CW) laser 147—is required to excite the components 160 in thefluid mixture (i.e., sperm cells). In one embodiment, the laser 147 (seeFIG. 3) emits a laser beam 148 through window 149 in layer 104, throughthe covering 133 at an uppermost portion of the chip 100, throughopening 150, and through covering 132 and window 152 in layer 101 of thechip 100, to illuminate the components 160 flowing through channel 164Bin interrogation region 129 of the chip 100.

In one embodiment, the light beam 148 can be delivered to the components160 by an optical fiber that is embedded in the microfluidic chip 100 atopening 150.

The high intensity beam 148 interacts with the components 160 (seedetailed explanation below), and passes through the first and secondcoverings 133, 132, to exit from bottom window 152, such that theemitted light 151, which is induced by the beam 148, is received by anobjective lens 153. The objective lens 153 may be disposed in anysuitable position with respect to the microfluidic chip 100. Because theinterrogation chamber 129 is sealed by the first and second coverings133, 132, the high intensity beam 148 does not impinge on themicrofluidic chip 100 and damage the layers 101-104. Thus, the first andsecond coverings 133, 132 help prevent damage to the microfluidic chip100 from the high intensity beam 148 and photonic noise induced from themicrofluidic chip material (i.e., plastic).

In one embodiment, the emitted light 151 received by the objective lens153 is converted into an electronic signal by an optical sensor 154,such as a photomultiplier tube (PMT) or photodiode, etc. The electronicsignal can be digitized by an analog-to-digital converter (ADC) 155 andsent to a digital signal processor (DSP) based controller 156. The DSPbased controller 156 monitors the electronic signal and may then triggerone of two actuator drivers (i.e., 157 a, 157 b) at a predeterminedvalue, to drive a relevant one of the two piezoelectric actuatorassemblies (109, 110, or 209,210). In one embodiment (shown in FIG. 2A),the piezoelectric drivers and piezoelectric actuators (158 a, 158 b, or219,220) are part of two piezoelectric actuator assemblies (109, 110, or209,210) respectively, disposed on either side of the interrogationchamber 129. The trigger signal sent to the piezoelectric actuators(109,110, or 219,220) is determined by the sensor raw signal, toactivate the particular piezoelectric actuator assembly (109,110,209,210) when the selected component is detected.

In the embodiment with the bonded piezoelectric actuator assemblies 109,110, the thickness of the diaphragm 170, 171 may be different and isdependent upon the voltage applied via electrical wires through theactuator assembly 1 09, 110 on the chip 100. When the electronic signalis sent through an electronic circuit directly to the actuatorassemblies (i.e., 109, 110), the diaphragms 170, 171 bend and change(increase) the pressure in the chambers 130, 131.

The at least one of the piezoelectric actuator assemblies (109, 110, or209,210) is used to act upon the desired components 160 in the fluidmixture in channel 164C, as the components 160 leave the opening 150 forinterrogation area 129 after interrogation. Although actuator driver 157b and piezoelectric actuator assembly 110 are not illustrated in FIG. 4,the operation and configuration of actuator driver 157 b andpiezoelectric actuator assembly 110 are the same as that of the actuatordriver 157 a and the piezoelectric actuator assembly 109. Thus,piezoelectric actuator 157 b acts to deflect components 160 in the flowstream in channel 164C to the right output channel 142, and to the thirdoutput 113. The same operation applies for the piezoelectric actuatorassembly 110, which jets sheath or buffer fluid 163 from the jet chamber131 via jet channel 128, and deflects target or selected components 160to the left output channel 140 and the third output 113.

In an alternative embodiment, a piezoelectric actuator assembly 106A(i.e., a piezoelectric disc similar to the piezoelectric actuatorassemblies 109, 110, and of a suitable size—see FIG. 2C), or a suitablepumping system (see FIG. 9, for example—discussed later), is used topump sample fluid 120 in channel 164 toward intersection 161. The samplepiezoelectric actuator assembly 1 06A would be disposed at sample input106. By pumping the sample fluid mixture 120 into the main channel 164,a measure of control can be made over the spacing of the components 160therein, such that a more controlled relationship may be made betweenthe components 160 as they enter the main channel 164.

If the piezoelectric actuator assemblies 109, 110 are not employed, the(target) components 160 proceed from main channel 164 to the centeroutput channel 141, and to the second output 112, and the sheath orbuffer fluids 163 proceed through output channels 140, 142, to outputs110, 112, respectively.

In one embodiment, the output channels 140-142 increase in dimensionfrom the channel 164C, leaving the interrogation chamber 129, such thatthe output ratio for enrichment of the isolated component 160, isincreased through the relevant channel(s).

Chip Operation

In one embodiment, the microfluidic chip 100 is provided in a sterilestate, and may be primed with one or more solutions (i.e., sheath orbuffer fluids 163), or purged of any fluids or materials by eitherdraining the microfluidic chip 100 or by flowing sheath or buffer fluids153 or other solutions through the microfluidic chip 100, according toknown methods. Once the microfluidic chip 100 is primed, and the jetchambers 130, 131 are filled with sheath or buffer fluids 163, eitherduring manufacturing, or thereafter (as described above), the air vents121, 122 are sealed. As stated above, in another embodiment, the airvents 121, 122 may be left open for additional sheath or buffer fluids163 to be added to the chambers 130, 131 during operation.

In one embodiment, as stated above, the components 160 that are to beisolated include, for example: isolating viable and motile sperm fromnon-viable or non-motile sperm; isolating sperm by gender, and other sexsorting variations; isolating stems cells from cells in a population;isolating one or more labeled cells from un-labeled cells distinguishingdesirable/undesirable traits; sperm cells with different desirablecharacteristics; isolating genes in nuclear DNA according to a specifiedcharacteristic; isolating cells based on surface markers; isolatingcells based on membrane integrity (viability), potential or predictedreproductive status (fertility), ability to survive freezing, etc.;isolating cells from contaminants or debris; isolating healthy cellsfrom damaged cells (i.e., cancerous cells) (as in bone marrowextractions); red blood cells from white blood cells and platelets in aplasma mixture; and isolating any cells from any other cellularcomponents, into corresponding fractions; damaged cells, or contaminantsor debris, or any other biological materials that are desired toisolated. The components 160 may be cells or beads treated or coatedwith, linker molecules, or embedded with a fluorescent or luminescentlabel molecule(s). The components 160 may have a variety of physical orchemical attributes, such as size, shape, materials, texture, etc.

In one embodiment, a heterogeneous population of components 160 may bemeasured simultaneously, with each component 160 being examined fordifferent quantities or regimes in similar quantities (e.g., multiplexedmeasurements), or the components 160 may be examined and distinguishedbased on a label (e.g., fluorescent), image (due to size, shape,different absorption, scattering, fluorescence, luminescencecharacteristics, fluorescence or luminescence emission profiles,fluorescent or luminescent decay lifetime), and/or particle positionetc.

In one embodiment, a two-step focusing method of a component sortingsystem consistent with the present invention may be used, as illustratedin FIG. SA, in order to position the components 160 in channel 164B forinterrogation in the interrogation chamber 129.

In one embodiment, the first focusing step of the present invention isaccomplished by inputting a fluid sample 120 containing components 160,such as sperm cells etc., through sample input 106, and inputting sheathor buffer fluids 163 through sheath or buffer inputs 107, 108. In oneembodiment, the components 160 are pre-stained with dye (e.g., Hoechstdye), in order to allow fluorescence, and for imaging to be detected. Inone embodiment, sheath or buffer fluids 163 are disposed in jet chambers130, 131, and inputs 121, 122 sealed.

In one embodiment, as shown in FIG. SA, components 160 in the samplefluid mixture 120 flow through main channel 164, and have randomorientation and position (see inset A). At intersection 161, the samplemixture 120 flowing in main channel 164 is compressed by the sheath orbuffer fluids 163 from channels 114, 115, in a first direction (i.e., atleast horizontally, on at least both sides of the flow, if not all sidesdepending on where the main channel 164 enters the intersection 161),when the sheath or buffer fluids 163 meet with the sample mixture 120.As a result, the components 160 are focused around the center of thechannel 164, and may be compressed into a thin strip across the depth ofthe channel 164A. The intersection 161 leading into channel 164A is thefocusing region. Thus, at intersection 161, as the sample 120 is beingcompressed by the sheath or buffer fluids 163 from channels 114, 115,toward the center of the channel 164A, the components 160 (i.e., spermcells) move toward the center of the channel 164 width.

In one embodiment, the present invention includes a second focusingstep, where the sample mixture 120 containing components 160, is furthercompressed by sheath or buffer fluids 163 from a second direction (i.e.,the vertical direction, from the top and the bottom) entering fromchannels 116, 117 at intersection 162. The intersection 162 leading intochannel 164B is the second focusing region. Note that although theentrances into intersection 162 from channels 116, 117 are shown asrectangular, one of ordinary skill in the art would appreciate that anyother suitable configuration (i.e., tapered, circular) may be used. Thesheath or buffer fluids 163 in the channels 116, 117 (which may bedisposed in different layers of the microfluidic chip 100 from channels164A-B) enter at different planes into the channel 164A-B, to align thecomponents 160 in the center of the channel 164B by both width and depth(i.e., horizontally and vertically) as they flow along channel 164 B.

Thus, in one embodiment, with the second focusing step of the presentinvention, the sample mixture 120 is again compressed by the verticalsheath or buffer fluids 163 entering at channels 116, 117, and thesample 120 stream is focused at the center of the channel 164B depth, asillustrated in FIG. SA, and the components 160 flow along the center ofthe channel 164B in approximately single file formation.

In one embodiment, the components 160 are sperm cells 160, and becauseof their pancake-type or flattened teardrop shaped head, the sperm cells160 will re-orient themselves in a predetermined direction as theyundergo the second focusing step—i.e., with their flat surfacesperpendicular to the direction of light beam 148 (see FIG. SA). Thus,the sperm cells 160 develop a preference on their body orientation whilepassing through the two-step focusing process. Specifically, the spermcells 160 tend to be more stable with their flat bodies perpendicular tothe direction of the compression. Hence, with the control of the sheathor buffer fluids 163, the sperm cells 160 which start with randomorientation, now achieve uniform orientation. Thus, the sperm cells 160not only make a single file formation at the center of the channel 164B,but they also achieve a uniform orientation with their flat surfacenormal to the direction of compression in the second focusing step.

Thus, all components 160 introduced into sample input 106, which may beother types of cells or other materials as described above, etc.,undergo the two-step focusing steps, which allow the components 160 tomove through the channel 164B in a single file formation, in a moreuniform orientation (depending on the type of components 160), whichallows for easier interrogation of the components 160.

In one embodiment, further downstream in channel 164B, the components160 are detected in the interrogation chamber 129 at opening 150 throughcoverings 132, 133, using the light source 147. Light source 147 emits alight beam 148 (which may be via an optical fiber) which is focused atthe center of the channel 164C at opening 150. In one embodiment, thecomponents 160, such as sperm cells 160, are oriented by the focusingstreams (i.e., sheath or buffer fluid 163 streams which act on samplestream 120) such that the flat surfaces of the components 160 are facingtoward the beam 148. In addition, all components 160 are moved intosingle file formation by focusing as they pass under beam 148. As thecomponents 160 pass under light source 147 and are acted upon by beam148, the components 160 emit the fluorescence which indicates thedesired components 160. For example, with respect to sperm cells, Xchromosome cells fluoresce at a different intensity from Y chromosomecells; or cells carrying one trait may fluoresce in a differentintensity or wavelength from cells carrying a different set of traits.In addition, the components 160 can be viewed for shape, size, or anyother distinguishing indicators.

In the embodiment of beam-induced fluorescence, the emitted light beam151 (in FIG. 3) is then collected by the objective lens 153, andsubsequently converted to an electronic signal by the optical sensor154. The electronic signal is then digitized by an analog-digitalconverter (ADC) 155 and sent to an electronic controller 156 for signalprocessing. The electronic controller can be any electronic processerwith adequate processing power, such as a DSP, a Micro Controller Unit(MCU), a Field Programmable Gate Array (FPGA), or even a CentralProcessing Unit (CPU). In one embodiment, the DSP-based controller 156monitors the electronic signal and may then trigger at least oneactuator driver (i.e., 157 a or 157 b), to drive one of the twopiezoelectric actuator assemblies (109, 110, or 219,220—part of therespective piezoelectric actuator assemblies 109, 110,209,210) when adesired component 160 is detected. In another embodiment, the FPGA-basedcontroller monitors the electronic signal and then either communicateswith the DSP controller or acts independently to trigger at least oneactuator driver (i.e., 157 a or 157 b), to drive one of the twopiezoelectric actuator assemblies (109,110, or 219,220—part of therespective piezoelectric actuator assemblies 109,110,209,210) when adesired component 160 is detected.

Thus, in one embodiment, selected or desired components 160 in channel164C in the interrogation chamber 129, are isolated by a jet stream ofbuffer or sheath fluids 163 from one of the jet channels 127, 128,depending on which output channel 140, 142 is desired for the selectedcomponent 160. In one exemplary embodiment, the electronic signalactivates the driver to trigger external stacked piezoelectric actuator219 (or activates driver 157 a to trigger actuator 109), at the momentwhen the target or selected component 160 arrives at the cross-sectionpoint of the jet channels 127, 128 and the main channel 164C. Thiscauses external stacked piezoelectric actuator assembly 209 (or 109) tocontact the diaphragm 170 and push it, compressing jet chamber 130, andsqueezing a strong jet of buffer or sheath fluids 163 from jet chamber130 via jet channel 127, into the main channel 164C, which pushes theselected or desired component 160 into output channel 142. Note that,similarly to the performance of stacked external piezoelectric actuatorassembly 209, the triggering of piezoelectric actuator assembly 210 (or110), would push a desired component 160 into the output channel 140 onthe opposite side from the jet 128.

Thus, sheath or buffer fluids 163 jetted from one of the jet channels127, 128 divert target or selected components 160 from their ordinarypaths in channel 164C, toward one of the selected or desired, respectiveoutput channels 140, 142, isolating those target components 160, andenriching the flows in those output channels 140, 142, and depleting theflow in the sample fluid 120 which continues straight out through outputchannel 141 with unselected components, if any. Thus, no triggering ofthe piezoelectric actuator assemblies 109, 110, means that theunselected components 160 in the fluid mixture 120 continue straight outthrough output channel 141.

In one embodiment, the isolated components 160 are collected from one ofthe first output 111, or the third output 113, using known methods inthe art, for storing, for further separation, or for processing, such ascryopreservation. Of course, components 160 that were not isolated intooutputs 111, 113, may also be collected from second output 112. Portionsof the first, second, and third outputs 111-113 may be characterizedelectronically, to detect concentrations of components, pH measuring,cell counts, electrolyte concentration, etc.

In one embodiment, interrogation of the sample 120 containing components160 (i.e., biological material), is accomplished by other methods. Thus,portions of, or outputs from, the microfluidic chip 100 may be inspectedoptically or visually. Overall, methods for interrogation may includedirect visual imaging, such as with a camera, and may utilize directbright-light imaging or fluorescent imaging; or, more sophisticatedtechniques may be used such as spectroscopy, transmission spectroscopy,spectral imaging, or scattering such as dynamic light scattering ordiffusive wave spectroscopy.

In some cases, the optical interrogation region 129 may be used inconjunction with additives, such as chemicals which bind to or affectcomponents of the sample mixture 120 or beads which are functionalizedto bind and/or fluoresce in the presence of certain materials ordiseases. These techniques may be used to measure cell concentrations,to detect disease, or to detect other parameters which characterize thecomponents 160.

However, in another embodiment, if fluorescence is not used, thenpolarized light back scattering methods may also be used. Usingspectroscopic methods, the components 160 are interrogated as describedabove. The spectrum of those components 160 which had positive resultsand fluoresced (i.e., those components 160 which reacted with a label)are identified for separation by the activation of the piezoelectricassemblies 109, 110, 209, 210.

In one embodiment, the components 160 may be identified based on thereaction or binding of the components with additives or sheath or bufferfluids 163, or by using the natural fluorescence of the components 160,or the fluorescence of a substance associated with the component 160, asan identity tag or background tag, or met a selected size, dimension, orsurface feature, etc., are selected for separation.

In one embodiment, upon completion of an assay, selection may be made,via computer 182 (which monitors the electronic signal and triggers thepiezoelectric assemblies 109, 110, 209,210) and/or operator, of whichcomponents 160 to discard and which to collect.

In one embodiment, after selection is made, a focused energy device mayemit a focused energy beam which damages or kills the selectedcomponents in the sample fluid mixture. The focused energy beam in suchan instance could be a laser effective to damage the selectedcomponents, such as cells. In a similar embodiment, after the damagingand killing by the focused energy beam, the selected and unselectedcomponents exit the microfluidic chip and are gathered in the same pool.

In one embodiment, the user interface of the computer system 182includes a computer screen which displays the components 160 in a fieldof view acquired by a CCD camera 183 over the microfluidic chip 100.

In one embodiment, the computer 182 controls any external devices suchas pumps (i.e., pumping mechanism of FIG. 9), if used, to pump anysample fluids 120, sheath or buffer fluids 163 into the microfluidicchip 100, and also controls any heating devices which set thetemperature of the fluids 120, 163 being inputted into the microfluidicchip 100.

Chip Cassette and Holder

The microfluidic chip 100 is loaded on a chip cassette 212, which ismounted on chip holder 200. The chip holder 200 is mounted to atranslation stage (not shown) to allow fine positioning of the holder200. The microfluidic chip holder 200 is configured to hold themicrofluidic chip 100 in a position such that the light beam 148 mayintercept the components 160 in the above described manner, at opening150. When the microfluidic chip 100 is in the closed position, a gasketlayer 105 (see FIG. 1) forms a substantially leak-free seal between themain body 211 and the microfluidic chip 100.

As illustrated in FIG. 6, in one embodiment, a microfluidic chip holder200 is made of a suitable material, such as aluminum alloy, or othersuitable metallic/polymer material, and includes a main body 211, and atleast one stacked external piezoelectric actuator 209,210.

The main body 211 of the holder 200 may be any suitable shape, but itsconfiguration depends on the layout of the chip 100. For example, thestacked external piezoelectric actuators 209,210 must be placed over thediaphragm(s) 170, 171, such that contact is made between a tip of thepiezoelectric actuator 219,220 and the diaphragm 170, 171 of themicrofluidic chip 100. The main body 211 of the holder 200 is configuredto receive and engage with external tubing (see FIG. 9) forcommunicating fluids/samples to the microfluidic chip 100.

The details of these cassette 212 and holder 200 and the mechanisms forattachment of the chip 100 to the cassette 212 and holder 200, are notdescribed in any detail, as one of ordinary skill in the art would knowthat these devices are well-known and may be of any configuration toaccommodate the microfluidic chip 100, as long as the objectives of thepresent invention are met.

As shown in FIG. 9, in one embodiment, a pumping mechanism includes asystem having a pressurized gas 235 which provides pressure for pumpingsample fluid mixture 120 from reservoir 233 (i.e., sample tube) intosample input 106 of the chip 100.

A collapsible container 237 having sheath or buffer fluid 163 therein,is disposed in a pressurized vessel 236, and the pressurized gas 235pushes fluid 163 to a manifold 238 having a plurality of differentoutputs, such that fluid 163 is delivered via tubing 231 a, 231 b to thesheath or buffer inputs 107, 108, respectively, of the chip 100.

A pressure regulator 234 regulates the pressure of gas 235 within thereservoir 233, and a pressure regulator 239 regulates the pressure ofgas 235 within the vessel 236. A mass flow regulator 232 a, 232 bcontrols the fluid 163 pumped via tubing 231 a, 231 b, respectively,into the sheath or buffer inputs 107, 108, respectively. Thus, tubing230, 231 a, 231 b is used in the initial loading of the fluids 120 intothe chip 100, and may be used throughout chip 100 to load sample fluid120 into sample input 106, or sheath or buffer inputs 107, 108. Inaddition, in one embodiment, tubing (not shown) can provide fluid 163from manifold 238 into air vents 121, 122 to fill chambers 130, 131, forexample.

In accordance with an illustrative embodiment, any of the operations,steps, control options, etc. may be implemented by instructions that arestored on a computer-readable medium such as a computer memory,database, etc. Upon execution of the instructions stored on thecomputer-readable medium, the instructions can cause a computing deviceto perform any of the operations, steps, control options, etc. describedherein.

The operations described in this specification may be implemented asoperations performed by a data processing apparatus or processingcircuit on data stored on one or more computer-readable storage devicesor received from other sources. A computer program (also known as aprogram, software, software application, script, or code) can be writtenin any form of programming language, including compiled or interpretedlanguages, declarative or procedural languages, and it can be deployedin any form, including as a stand-alone program or as a module,component, subroutine, object, or other unit suitable for use in acomputing environment. A computer program may, but need not, correspondto a file in a file system. A program can be stored in a portion of afile that holds other programs or data (e.g., one or more scripts storedin a markup language document), in a single file dedicated to theprogram in question, or in multiple coordinated files (e.g., files thatstore one or more modules, subprograms, or portions of code). A computerprogram can be deployed to be executed on one computer or on multiplecomputers that are located at one site or distributed across multiplesites and interconnected by a communication network. Processing circuitssuitable for the execution of a computer program include, by way ofexample, both general and special purpose microprocessors, and anyone ormore processors of any kind of digital computer.

It should be noted that the orientation of various elements may differaccording to other illustrative embodiments, and that such variationsare intended to be encompassed by the present disclosure.

The construction and arrangements of the microfluidic chip, as shown inthe various illustrative embodiments, are illustrative only. Althoughonly a few embodiments have been described in detail in this disclosure,many modifications are possible (e.g., variations in sizes, dimensions,structures, shapes and proportions of the various elements, values ofparameters, mounting arrangements, use of materials, colors,orientations, etc.) without materially departing from the novelteachings and advantages of the subject matter described herein. Someelements shown as integrally formed may be constructed of multiple partsor elements, the position of elements may be reversed or otherwisevaried, and the nature or number of discrete elements or positions maybe altered or varied. The order or sequence of any process, logicalalgorithm, or method steps may be varied or re-sequenced according toalternative embodiments. Other substitutions, modifications, changes andomissions may also be made in the design, operating conditions andarrangement of the various illustrative embodiments without departingfrom the scope of the present disclosure.

1-43. (canceled)
 44. An apparatus for identifying components in a fluidmixture, comprising: (i) a microfluidic chip, comprising: (a) a sampleinput channel disposed in a first structural layer, said sample inputchannel for receiving a fluid mixture containing a plurality ofcomponents, wherein said sample input channel comprises: (i) a firstintersection; and (ii) a second intersection; (b) a first plurality ofsheath fluid channels disposed in said first structural layer, whereinsaid first plurality of sheath fluid channels intersect said sampleinput channel at said first intersection; (c) a second plurality ofsheath fluid channels disposed in a second structural layer, whereinsaid second plurality of sheath fluid channels intersect said sampleinput channel at said second intersection; and (d) a plurality of outputchannels fluidly connected to and branching from said sample inputchannel, wherein each of said plurality of output channels is disposedbetween a pair of recessed portions at an end of said first and secondstructural layers; (ii) an interrogation apparatus disposed downstreamof said second intersection, said interrogation apparatus whichdifferentiates said plurality of components into selected components andunselected components; and (iii) a focused energy device that acts uponsaid selected components.
 45. The apparatus of claim 44, wherein saidsample input channel tapers at an entry point into said firstintersection such that sheath fluids from said first plurality of sheathfluid channels compress said plurality of components in said fluidmixture on at least two sides.
 46. The apparatus of claim 45, whereinsheath fluids from said first plurality of sheath fluid channelscompress said plurality of components in said fluid mixture on allsides.
 47. The apparatus of claim 44, wherein said sample input channeltapers into said interrogation apparatus to increase the velocity ofsaid fluid mixture traveling through said interrogation apparatus. 48.The apparatus of claim 44, wherein said focused energy device emits afocused energy beam to damage selected components.
 49. The apparatus ofclaim 44, wherein said interrogation apparatus comprises a light sourcethat emits a light beam to illuminate and excite said plurality ofcomponents in said fluid mixture.
 50. The apparatus of claim 44, furthercomprising: a pumping mechanism that pumps said fluid mixture into saidsample input channel and sheath fluid into said first and secondplurality of sheath fluid channels.
 51. The apparatus of claim 44,wherein said plurality of components are a plurality of cells.
 52. Theapparatus of claim 51, wherein said focused energy device emits afocused energy beam to kill selected cells.
 53. The apparatus of claim52, wherein said interrogation apparatus interrogates said plurality ofcells to differentiate selected cells based on viability, motility,gender, label, desirable trait, DNA content, surface marker, membraneintegrity, predicted reproductive status, health, or survivalcharacteristics.
 54. The apparatus of claim 53, wherein said pluralityof cells are sperm.
 55. An apparatus for generating a fluid mixture oflive and killed cells, comprising: (i) a microfluidic chip, comprising:(a) a sample input channel disposed in a first structural layer, saidsample input channel for receiving a fluid mixture containing aplurality of cells, wherein said sample input channel comprises a firstintersection; (b) a first plurality of sheath fluid channels disposed insaid first structural layer, wherein said first plurality of sheathfluid channels intersect said sample input channel at said firstintersection; wherein said sample input channel tapers at an entry pointinto said first intersection such that said plurality of cells in saidfluid mixture are vertically and horizontally compressed by sheath fluidfrom said first plurality of sheath fluid channels; (c) at least oneoutput channel fluidly connected to said sample input channel; (ii) aninterrogation apparatus that interrogates said plurality of cells toselect cells to be killed; and (iii) a focused energy device that killssaid selected cells, wherein said at least one output channel isdisposed downstream of said focused energy device to receive said fluidmixture of live and killed cells.
 56. The apparatus of claim 55, whereinsaid microfluidic chip further comprises: (d) a second plurality ofsheath fluid channels disposed in a second structural layer, said secondplurality of sheath fluid channels that intersect said sample inputchannel at a second intersection between said first intersection andsaid interrogation apparatus.
 57. The apparatus of claim 56, whereinsheath fluids from said second plurality of sheath fluid channelsfurther vertically compress said plurality of cells in said fluidmixture.
 58. The apparatus of claim 55, wherein said interrogationapparatus comprises a light source that emits a light beam to illuminateand excite said plurality of cells in said fluid mixture.
 59. Theapparatus of claim 56, further comprising: a pumping mechanism thatpumps said fluid mixture into said sample input channel and sheath fluidinto said first and second plurality of sheath fluid channels.
 60. Theapparatus of claim 55, wherein said interrogation apparatus interrogatessaid plurality of cells to select cells based on viability, motility,gender, label, desirable trait, DNA content, surface marker, membraneintegrity, predicted reproductive status, health, or survivalcharacteristics.
 61. The apparatus of claim 55, wherein said pluralityof cells are sperm.
 62. The apparatus of claim 61, wherein saidinterrogation apparatus interrogates said sperm to select cells based ongender.